RRC ID 33479
Author Masai H, Arai K.
Title Leading strand synthesis of R1 plasmid replication in vitro is primed by primase alone at a specific site downstream of oriR.
Journal J Biol Chem
Abstract By using an in vitro system for R1 plasmid replication dependent on a plasmid-encoded repA protein and host dnaA protein, 5' ends of the nascent leading strand were located at positions 1986-1992, some 380 base pair downstream of oriR. Analyses of early replication intermediates generated in vitro in the presence of dideoxy TTP also indicated that replication initiates about 400 base pair downstream of oriR and proceeds unidirectionally. When a 418-base single-stranded DNA from position 1778 to 2195, derived from the leading strand template, was cloned onto an M13 vector, the chimeric single-stranded phage could be replicated in vitro with only single-stranded DNA binding protein, primase (dnaG gene product), and DNA polymerase III holoenzyme. Furthermore, the priming occurred at a site identical to leading strand initiation. These results strongly suggest that the leading strand synthesis is primed by primase alone. The lagging strand synthesis is specifically terminated at position 1515 or 1516 within oriR, preventing further leftward fork movement. Based on these results, a scheme of R1 plasmid replication is presented.
Volume 264(14)
Pages 8082-90
Published 1989-5-15
PII S0021-9258(18)83153-2
PMID 2542261
MeSH Bacterial Proteins / genetics Bacterial Proteins / physiology Bacteriophage phi X 174 / genetics Bacteriophages / genetics Base Sequence Cloning, Molecular DNA Polymerase III / metabolism DNA Primase DNA Replication* DNA Restriction Enzymes DNA, Single-Stranded / biosynthesis* DNA, Viral / biosynthesis DNA-Binding Proteins / metabolism Dideoxynucleotides Endonucleases / metabolism Escherichia coli / genetics* Nucleic Acid Hybridization Plasmids* RNA Nucleotidyltransferases / metabolism* Single-Strand Specific DNA and RNA Endonucleases Templates, Genetic Thymine Nucleotides / metabolism
IF 4.238
Times Cited 45
Prokaryotes E. coli pHM6050